EcoRI contaminated (so use less)

HARDIES at THORIN.UTHSCSA.EDU HARDIES at THORIN.UTHSCSA.EDU
Wed Nov 23 10:08:15 EST 1994


Padmaja Mandalaparthy writes:
> ...the Eco R1 enzyme and
> buffer we have seem to be contaminated leading to DNA degradation problems
> repeatedly in our lab. We are using absolutely sterile tips, tubes and
> distilled water.... Even the brand new stuff is
> degrading absolutely good batches of both plasmid and lambda gt10 dna
> preps. 

Check to see that you're not just using too much enzyme.  Most manufacturers
don't quarantee that it's cleaner than enough to get by with 100x excess.
So, for example, if you put in 1 ul (maybe 20 U) for 0.1 ug 
and digest 3 hours, you've used a 600 x excess and exceeded
the margin of safety.  I recommend sticking to 5-10x excess unless
you're actually seeing trouble cutting.

Steve Hardies, Dept. of Biochemistry, Univ. of Texas HSC at San Antonio
Hardies at thorin.uthscsa.edu





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